The control of plant morphology is of major importance in the commercial production of plants for agricultural or horticultural purposes, to enhance productivity and yield, to improve the efficiency of husbandry and harvest, and to achieve aesthetic desirability. Features which require control or modification may include the morphology of the flower, fruit or tuber, the quantity of flowers, fruit, seed or tubers, the extent of primary and lateral roots, the form of the aerial shoots or trunk, and the presence of thorns or stinging hairs. Other features which may be desirably controlled include the advancement or delay of abscission of leaves, flowers or fruit, the release of seeds, and the production of storage organs or secretory glands.
Morphological changes often occur as a result of environmental impact on the plant, including physical damage, herbivore predation, pathogen infection, cold, heat, and drought. They can often be brought about deliberately by human intervention, either physically (pruning, bending, tying, staking, or excising particular organs or structures) or chemically (application of agrochemicals and plant growth substances). Whichever is the causative agent, morphological changes are enacted by expression of genes within the cells of the plant itself. At the onset of the change, the initiation of expression of one or more genes occurs in those particular tissues where cell growth, proliferation, development or necrosis is required to culminate in the gross physical change.
The expression of a gene is dependent upon its DNA sequence being transcribed into RNA by the action of RNA polymerase. To achieve this, RNA polymerase must recognise and attach to a region of DNA sequence located upstream of (i.e. 5′ to) the gene coding sequence in order for transcription to be initiated. Such a region is termed the promoter of the gene. The intrinsic nature of the promoter sequence determines the circumstances and the manner in which the gene is expressed.
There are, broadly speaking, four types of promoters found in plant tissues; constitutive, tissue-specific, developmentally-regulated, and inducible/repressible, although it should be understood that these types are not necessarily mutually exclusive.
A constitutive promoter directs the expression of a gene throughout the various parts of a plant continuously during plant development, although the gene may not be expressed at the same level in all cell types. Examples of known constitutive promoters include those associated with the cauliflower mosaic virus 35S transcript (Odell et al, 1985), the rice actin 1 gene (Zhang et al, 1991) and the maize ubiquitin 1 gene (Cornejo et al, 1993).
A tissue-specific promoter is one which directs the expression of a gene in one (or a few) parts of a plant, usually throughout the lifetime of those plant parts. The category of tissue-specific promoter commonly also includes promoters whose specificity is not absolute, i.e. they may also direct expression at a lower level in tissues other than the preferred tissue. Examples of tissue-specific promoters known in the art include those associated with the patatin gene expressed in potato tuber and the high molecular weight glutenin gene expressed in wheat, barley or maize endosperm.
A developmentally-regulated promoter directs a change in the expression of a gene in one or more parts of a plant at a specific time during plant development. The gene may be expressed in that plant part at other times at a different (usually lower) level, and may also be expressed in other plant parts.
An inducible promoter is capable of directing the expression of a gene in response to an inducer. In the absence of the inducer the gene will not be expressed. The inducer may act directly upon the promoter sequence, or may act by counteracting the effect of a repressor molecule. The inducer may be a chemical agent such as a metabolite, a protein, a growth regulator, or a toxic element, a physiological stress such as heat, wounding, or osmotic pressure, or an indirect consequence of the action of a pathogen or pest. A developmentally-regulated promoter might be described as a specific type of inducible promoter responding to an endogenous inducer produced by the plant or to an environmental stimulus at a particular point in the life cycle of the plant. Examples of known inducible promoters include those associated with wound response, such as described by Warner et at (1993), temperature response as disclosed by Benfey & Chua (1989), and chemically induced, as described by Gatz (1995).
A promoter sequence may comprise a number of defined domains necessary for its function. A first of these comprises approximately 70 base pairs located immediately upstream of (that is, 5′ to) the structural gene and forms the core promoter. The core promoter contains the CAAT and TATA boxes and defines the transcription initiation site for the gene. A series of regulatory sequences upstream of the core promoter constitute the remainder of the promoter sequence and determine the expression levels, the spatial and temporal patterns of expression, and the response to inducers. In addition some promoters contain sequence elements which act to enhance the level of expression, for example that from the pea plastocyanin promoter as described in International Patent Publication No. WO 97/20056.
Genetic modification of plants depends upon the introduction of chimaeric genes into plant cells and their controlled expression under the direction of a promoter. Promoters may be obtained from different sources including animals, plants, fungi, bacteria, and viruses, and different promoters may work with different efficiencies in different tissues. Promoters may also be constructed synthetically.
It may often be desirable to express introduced genes in a number of different tissues within a plant. For example the expression of a resistance to a pathogen or pest, or tolerance to temperature extremes might be best expressed throughout all tissues in a plant. Similarly it might be desirable to ensure the expression of the transgenes at all times throughout the development of the plant. Also, a promoter which is expressed in a manner that is immune to the influence of inducers or repressors resulting from unforeseen environmental stimuli may also be useful to ensure the continued expression of a trait. For these purposes, the use of a “constitutive” promoter would be desirable. Examples of constitutive promoters include the CaMV 35S promoter. For cereals the ubiquitin promoter is a constitutive promoter of choice (Christensen & Quail, 1996).
However, in some instances it is more desirable to control the location of gene expression in a transgenic plant. This may enhance the effect of gene expression by ensuring that expression occurs preferentially in those tissues where the effect of the gene product is most efficacious. By the same argument, modulated expression can reduce potential yield loss by limiting the resource drain on the plant. Further advantages include limiting the expression of agronomically useful yet generally deleterious genes to specific tissues by localisation and compartmentalisation of gene expression in cases where the gene product must be restricted to, or excluded from, certain tissues. For example, anther specific expression of the suc inhibitor genes (Mariani et al, 1990) has been used in male sterility systems, whereas expression in other parts of the plant would result in toxicity. A similar cell death system is described in International Patent Application WO 89/10396 where an RNAse protein is used in combination with an anther specific promoter to cause necrosis of the anther cells and confer male sterility on the plant.
In International Patent Applications WO 02/33106 and WO 02/33107 are described plant cell death systems providing resistance to nematode infection by the expression of a ribosome inactivating protein (Maize Ribosome Inactivating Protein, Pokeweed Antiviral Protein) under the regulation of nematode feeding site specific promoters. In these cases the specificity of expression of the deleterious gene is enhanced by the promoters being both tissue specific and responsive to nematode invasion.
In some instances two or more transgenes may be expressed in a plant in similar or different locations. Each transgene may be expressed under the control of a different promoter which expresses in more than one region of the plant. The promoters may be selected so that there is an overlap in their respective expression sites at one or more desired locations. This overlap site(s) gives increased specificity and targeting of gene expression. By judicious selection of the gene product encoded by each transgene, the overlapping expressing of both transgenes may lead to an additive or enhanced effect on the target tissues, whereas expression of only one or other of the transgenes at other locations may cause no effect on the plant. For example in International Patent Application WO 02/33106 two separate peptide domains derived from Maize Ribosomal Inhibitory Protein (RIP) are expressed under the regulation of two different tissue specific promoters, having different expression profiles but which nevertheless have one site in common, resulting in the production of an active protein at the site of overlap.
Conversely, the two transgenes may encode an effector molecule and an agonist molecule or protectant. In this case, the effector molecule will affect the plant at all locations where it is expressed, except those where the expression site overlaps with that of expression of the agonist or protectant molecule. In NZ 260511 a plant cell death system is proposed with increased tissue specificity. This system comprises the expression of a cytotoxic molecule (under the control of a first promoter, which first promoter causes expression in specific target cells and at one or more other sites in the plant), in conjunction with a protective molecule (under the control of a second promoter, which second promoter causes expression in all of the sites where the first promoter is active except the specific target cells). Examples of suitable cytotoxic and protective molecules are proteases and protease inhibitors, respectively, or nucleases and nuclease inhibitors, respectively. WO 93/10251 discloses the use of a cytotoxic ribonuclease molecule Barnase together with the protective inhibitor molecule Barstar.
Another example of a two-component transgenic system is provided in International Patent Application No. WO98/44138. This system comprises the expression of a gene product under the control of a promoter, which promoter and gene product are selected so that there is an overlap in their respective expression and effector sites at a desired location. The promoter directs expression in the specific cells and also at one or more other sites in the plant, whilst the molecular target of the gene product occurs in a second range of cells also including the specific target cells. This overlap site(s) gives increased specificity and targeting of gene expression to the cells at the desired location. By judicious selection of the gene product encoded by the transgene, the expression in non-target cells causes no effect on the plant.
A major application of the localised expression of a deleterious gene to a particular tissue, would be in the modification of plant morphology, for example by the controlled necrosis or prevention of development of certain tissues or organs, such as flowering structures, fruiting bodies, storage tissues, shoots, leaf tissues, root tissues, abscission zones, secretory glands, stinging cells, trichomes, or thorns.
A particular application of localised expression of a deleterious gene to modify plant morphology would be in the prevention of lateral shoot outgrowths from leaf axillary meristems. The anatomy of axillary meristems and lateral buds is described in Esau (1960). Outgrowth of lateral shoots most commonly arises when the dominance of the apical shoot is removed or reduced; for example when the apical shoot is damaged or removed, either accidentally through physical damage or predation by herbivores, or as part of agricultural practice e.g. coppicing. Other changes which modify for example the production, transport, detection, or metabolism of endogenous plant growth substances may also cause outgrowth from axillary meristems. Lateral shoots, or “suckers”, may be undesirable for purely aesthetic reasons, may produce a plant with unusable morphology, or may have a detrimental metabolic effect on the plant as a whole by acting as an additional source or sink for various metabolites or plant growth substances.
One example where lateral bud outgrowth occurs is in the commercial cultivation of tobacco, where the apical shoot comprising the inflorescence and uppermost leaves is removed at a specific time during the growth of the plant, in a process named “topping”, to stimulate growth and development of the remaining leaves, to enhance root growth, and to encourage the redistribution of metabolites and secondary compounds to the plant leaves. A drawback to the topping process is that it also stimulates the outgrowth of lateral shoots which thereby offsets the desired redistribution of metabolites. This effect is commonly overcome by the physical removal of the lateral shoots which is highly labour intensive or by the application of chemical shoot suppressants such as maleic hydrazide, which is both costly in terms of materials and may result in the retention of chemical residues on the harvested plant. A system which prevents such “suckering” by specifically directing the disruption of those cells involved in lateral bud outgrowth, would therefore provide a great benefit to the cultivation of tobacco.
Ribosome inactivating proteins (RIPs) are a group of toxic plant proteins that catalytically inactivate eukaryotic ribosomes (Stirpe and Barbieri 1986). RIPs function as N-glycosidases to remove a specific adenine in a conserved loop of the large rRNA, and thereby prevent binding of Elongation Factor 2, thus blocking cellular protein synthesis. Three forms of RIPs have been described. Type 1 RIPs such as pokeweed antiviral protein and barley translation inhibitor are each comprised of a single polypeptide chain, each with an approximate Mr value of 30,000. Type 2 RIPs such as ricin, abrin and modeccin each comprise two polypeptide chains, one with RIP activity linked by a disulphide bond to the other galactose-binding lectin chain. Type 3 RIPs such as maize RIP comprise a single polypeptide chain which subsequently undergoes proteolytic cleavage to release two active peptide domains.
Pokeweed (Phytolacca americana) produces three distinct antiviral proteins, namely PAP′, PAPII and PAP-S that appear in spring leaves, summer leaves and seeds, respectively. Amino acid similarities between these three proteins have been observed. As used herein ‘PAP’ covers all three of these antiviral proteins.
U.S. Pat. No. 6,015,940 discloses the preparation of a cDNA clone of PAP′ prepared from spring leaves of pokeweed, and the use thereof under the control of a constitutive promoter (either cauliflower mosaic virus 35S promoter or the figwort mosaic virus 35S promoter) in the production of transgenic tobacco and potato plants resistant to infection by the viruses PVX and PVY.
Transgenic plants containing the summer leaf form of PAP, PAP-II, have been described in WO 99/60843. A number of full length and truncated PAP-II gene sequences were screened in order to identify those variant PAP-II proteins which retained antiviral activity but exhibited no phytotoxicity. Transgenic plants exhibited both antiviral and antifungal activity.
The PAP gene is expressed in vivo in leaves initially to produce an inactive Pro-PAP protein. It is known that following translation, the Pro-PAP′ protein molecule is targeted to the cell wall. At some stage during this process the N- and C-terminal extensions of the Pro-PAP′ molecule are cleaved to produce an activated PAP′ molecule (mature PAP′). In the case of PAP-S (expressed in seeds) the cellular localisation is not known. However, the N-terminal processed region of PAP-S appears to have properties similar to signal sequences for targeting.
The structure of the mature PAP-S protein, i.e. with N- and C-terminal extensions removed, may be described in terms of two separate domains, corresponding to the two domains of Type 3 RIPs, or the two polypeptides of Type 2 RIPs, i.e. the ribosome binding domain and the catalytic domain.